Selectivity is an important factor for a successful chromatographic separation. Common stationary phases for liquid chromatography, such as reversed-phase (RP), ion-exchange (IEX) and normal phase (NP) chromatography are frequently characterized by limited selectivity necessitating multiple analyses for a single sample.
Reversed-phase (RP) stationary phases for liquid chromatography (LC) are both popular and commercially available. In recent years, stationary phases containing a phenyl type functionality have been introduced and are widely used. A recent report indicates that 35% of all RPLC analyses employed C18 phases and 19% used phenyl-type phases (Ronald E. Majors, Trends in HPLC Column Usage, LCGC North America, Nov. 1, 2009, pp 956-972). The increased popularity of phenyl phases results from their unique chromatographic selectivity complimentary to alkyl-type phases (e.g., C18) in method developments because of their capacity to participate in aromatic interaction with aromatic solutes.
Phenyl bonded phases have been successfully used to resolve positional isomers (L. Zhou, Y. Wu, B. D. Johnson, J. M. Wyvratt, J. Chromatogr. A 866 (2000), p. 281), tocopherols (S. L. Richhemier, M. C. Kent, M. W. Bernat, J. Chromatogr. A 677 (1994), p. 75), flavonoids (F. Dondi, Y. D. Kahie, J. Chromatogr. 461 (1989), p. 281), taxols (D. C. Locke, R. Dolfinger, Anal. Chem. 75 (2003), p. 1355), polynuclear aromatics and nitroaromatic compounds (D. H. Marchand, K. Croes, J. W. Dolan, L. R. Snyder, R. A. Henry, K. M. R. Kallury, S. Waite, P. W. Carr, J. Chromatogr. A 1062 (2005), p. 65), active pharmaceutical ingredient and related compounds (D. H. Marchand, K. Croes, J. W. Dolan, L. R. Snyder, R. A. Henry, K. M. R. Kallury, S. Waite, P. W. Carr, J. Chromatogr. A 1062 (2005), p. 65).
Although there are a number of commercial phenyl-type phases in the market, all of them have at least one of the following drawbacks that prevent them from broad application: low hydrophobic retention, inadequate shape selectivity for analytes, and incompatibility with highly aqueous mobile phases. The most common commercial phenyl columns contain a phenyl ligand with a short alkyl linker covalently bound to the silica surface, however, due to the short alkyl linker, these columns usually lack sufficient hydrophobic retention and exhibit low hydrolytic stability.
A great deal of research has been done to prepare stationary phases containing various aromatic groups, such as phenyl (J. D. Goss, J. Chromatogr. A 828 (1998), p. 267), pyrenyl (K. Kimata, T. Hirose, K. Mariachi, K. Kosoya, T. Araki, N. Tanaka, Anal. Chem. 67 (1995), p. 2556 and N. Tanaka, Y. Okuda, K. Iwaguchi, M. Araki, J. Chromatogr. 239 (1982), p. 761), naphthenyl (N. Tanaka, Y. Okuda, K. Iwaguchi, M. Araki, J. Chromatogr. 239 (1982), p. 761), fluorenyl (R. R. Brindle, K. Albert, J. Chromatogr. A 757 (1997), p. 3), anthracenyl (C. Grosse-Rhode, H. G. Kicinski, A. Kettrup, Chromatographia 29 (1990), p. 489) and naphthalimide (J. Horak, N. M. Maier, W. Lindner, J. Chromatogr. A 1045 (2004), p. 43).
Nakashima et al. used a packing material, 3-(1,8-naphthalimido)propyl-modified silyl silica gel, as a stationary phase for high-performance liquid chromatography. It was reported that this material behaved like a reversed-phase stationary phase with some π-π interaction, and was used to separate purine derivatives, i.e., xanthine, hypoxanthine, uric acid, theobromine, theophylline and caffeine (K. Nakashima et al. J. Chromatogr. A 722 (1996), pp. 107-113). However, due to the short alkyl linker, hydrophobic retention is too low for broad application when a reverse phase modality is desired. In addition, the synthetic route used by Nakashima et al. involved a two-step reaction, resulting in undesirable π-π interaction or aromatic stacking interaction and an anion-exchange “mixed-mode” material. This synthetic route also provided only a low bonding density of the organic ligands, leading to low shape selectivity and low hydrolytic stability.
J. Horak et al. reported preparation and chromatographic evaluation data of three naphthalimide-type stationary phases (J. Horak et al. J. Chromatogr. A 1045 (2004), pp. 43-58). These phases consisted of a naphthalimide end-group to provide an aromatic stacking interaction and a long alkyl spacer with a thiol ether linkage. They reported that the presence of electron donor/acceptor moieties within a reversed phase system not only increased the overall retention times for aromatic solutes, but also led to an enhanced shape selectivity of the hybrid stationary phase. However, these phases have quite a few drawbacks. Firstly, thiol and thiol ether groups are not stable, and are subject to oxidation and/or cleavage under acidic conditions. Thus, they are not viable chromatographic packing materials. Secondly, the synthesis involves free radical addition between R1—SH and CH2═CH—R2, generating an undesirably complex mixed-mode surface, including an aromatic interaction moiety, a thiol ether and unreacted thiol groups. Furthermore, due to steric hindrance, the reported synthetic approach produces very low bonding density (1.08 to 1.73 μmol/m2). Low bonding density has adverse effects on chromatographic properties, including low shape selectivity, low hydrophobic retention and low hydrolytic stability
Most high surface coverage reversed-phase columns are not compatible with highly aqueous mobile phases, suffering from sudden retention time loss (a phenomenon often referred to as stationary phase de-wetting or phase collapse) after interrupting mobile phase flow. An exemplary solution to this problem is the use of polar-end-capping or the incorporation of polar groups, including amide, sulfonamide, carbamate, or urea groups in the alkyl chain. However, phenyl-type phases have lower hydrophobic retention compared to their alkyl-type counterpart with the same number of carbon atoms and adding a polar group further exacerbates this problem. Thus it's only by combining all three components (i.e., a longer, more hydrophobic linker, a polar group and an aromatic moiety fused to the polar group that a useful aromatic interaction stationary phase with reverse phase properties, which is compatible with highly aqueous mobile phases can be prepared. The present invention provides stationary phases having such properties.